An overall goal of this Center Grant is to carry out a safe and informative Phase I Clinical Trial. A critical component of this goal is assurance that the regulatory cassettes used for expressing therapeutic cDNAs be highly active in human skeletal muscle cells, so that beneficial levels of therapeutic proteins are produced, and silent in human non-muscle cells, so as to minimize the possibility of an immune response to the therapeutic protein. Our lab is responsible for designing and testing the regulatory cassettes which the Chamberlain lab will combine with therapeutic cDNAs and use in the construction of viral vectors. The major components of this proposal are: (1.) Modification of current MCK regulatory gene cassettes for optimal expression levels and tissue specificity based on quantitative studies in normal and dystrophic human and canine cell cultures. (2.) Development of immunodeficient mouse model test systems in which functionally mature normal and dystrophic human muscle tissue is reconstructed in mouse Tibialis anterior (fast) and Soleus (slow) muscles, and use of these models for the realistic testing of viral transduction and therapeutic protein expression in human muscle. (3.) Clonal analysis of the proliferative and developmental capacities of canine dystrophic muscle cells in conjunction with vector- and cell-mediated therapies. Our studies should assure that the regulatory gene cassettes used for expressing therapeutic cDNAs in human muscle disease patients have been subjected to rigorous pre-testing in the contexts of functional human and large animal muscle tissues, prior to their use in clinical trials. Our studies should also indicate whether more futuristic vector- or cell-mediated therapies can target or replace the normal regenerative satellite cell population. The latter goal is critical to successful long-term muscle gene therapy because muscle fibers - especially those in diseased muscle - undergo periodic regeneration.